Natural transformation in Streptococcus thermophilus : Regulation autolysis and ComS*-controlled gene expression.

Abstract

Natural genetic transformation has been extensively studied in Streptococcus pneumoniae for generations, and much has been learned about this important phenomenon since its discovery by Frederick Griffith in 1928. In comparison, natural transformation has been little studied in other streptococcal species. The majority of streptococci have, in fact, never been observed to be naturally transformable. Streptococcus thermophilus has traditionally been considered to be a non-competent species. It is widely used by the dairy industry in the production of a variety of food products, and is consequently of great economic importance. To further improve the properties of S. thermophilus as a dairy starter, a better understanding of its genetics, metabolism and physiology is essential. Progress in this area has been hampered by the lack of efficient genetic tools. Some years ago, Blomqvist and co-workers therefore set out to investigate whether S. thermophilus could be made competent for natural transformation by artificial overexpression of the alternative sigma factor ComX. This strategy proved successful resulting in a new tool that opened up new possibilities with respect to genetic manipulation of the S. thermophilus genome. Furthermore, the discovery sparked the interest of a number of research groups, leading to increased activity in this field. In the current study, an important goal was to better understand how expression of ComX, the master switch of competence induction in streptococci, is regulated in S. thermophilus. Our results show that ClpC, presumably in complex with ClpP, regulates the level of ComX in the bacterial cell post-transcriptionally. ClpC is not part of the quorumsensing-like competence-induction pathway recently identified by Fontaine and colleagues, but seems to be part of a control mechanism that prevents spontaneous competence induction in S. thermophilus under conditions that are sub-optimal or inappropriate for competence development (Paper I). In S. pneumoniae competent cells produce and secrete a murein hydrolase (CbpD-Sp) that kills and lyses non-competent pneumococci and members of related species. Evidence strongly indicates that the biological function of CbpD-Sp is to mediate release of homologous DNA from target cells that can be taken up by competent cells to serve as templates for recombinational DNA repair. In silico screening of the S. thermophilus genome showed that it encodes a CbpD-like protein (CbpD-St) with a unique C-terminal domain. Consequently, it was of interest to determine whether this protein carries out the same function in S. thermophilus as in S. pneumoniae. Our results showed that the properties of CbpD-St and CbpD-Sp are similar in most respects. Both proteins are murein hydrolases that bind to the surface of their respective host cells via their C-terminal domains. Both proteins also have the potential to lyse susceptible cells. However, in contrast to its pneumococcal counterpart, CbpD-St has a positive effect on the transformability of S. thermophilus (paper II). Thus, although our results indicate that CbpD-St and CbpD-Sp probably are functional analogues, it cannot be ruled out that CbpD-St serves a different biological function in S. thermophilus. While the present study was in progress, the sought-after quorum-sensing pathway controlling competence induction in S. thermophilus was identified by Fontaine et al. The pathway turned out to be completely unrelated to the corresponding quorum-sensing pathway in S. pneumoniae. This made us realize that the S. thermophilus pathway, in principle, could be developed into a peptide-regulated gene depletion system for use in S. pneumoniae. The pathway consists of a signalling peptide (ComS*), which is imported into the cytoplasm by the Ami oligopeptide transporter, and an intracellular transcriptional activator termed ComR. According to the model proposed by Fontaine et al., ComR becomes activated upon binding to ComS*. In the active state ComR induces expression of ComX by binding to the comX promoter. By introducing the comR gene and comX promoter fused to the gene of interest into the genome of S. pneumoniae we were able to show that the gene depletion system functioned as intended. The essential licD1gene, which is required for the synthesis of wall and lipoteichoic acids in S. pneumoniae, was used as a test case. Our results showed that depletion of the licD1 gene gives rise to oversized, elongated and misshapen cells, indicating that pneumococcal cells with low levels of teichoic acids struggle to divide. In sum, the ComRS-based gene depletion system described in paper III has excellent properties that should make it a very useful tool for the study of essential genes in S. pneumoniae and other Gram-positive bacteria.

Mens det foreliggende studiet pågikk ble den ovenfor nevnte ”quorum-sensing”- lignende signaloverføringsveien som induserer kompetanse hos S. thermophilus identifisert av Fontaine et al.. Det viste seg at denne signaloverføringsveien er totalt ubeslektet med den tidligere identifiserte signaloverføringsveien som benyttes til kompetanseinduksjon hos S. pneumoniae. Dette gav oss ideen til å forsøke å konstruere et titrerbart peptid-kontrollert genekspresjonssystem til studier av essensielle gener hos pneumokokker. Med et slikt system ville vi kunne uttrykke essensielle gener ektopisk, dvs. via det peptid-kontrollerte genekspresjonssystemet fra S. thermophilus. Etter delesjon av det native målgenet vil så den ektopiske transkripsjonen kunne senkes gradvis til fenotypiske trekk som gir informasjon om funksjonen til det essensielle genet manifesterer seg. Signaloverføringsveien som ble identifisert av Fontaine et al. består av et signalpeptid (ComS*), som transporteres inn i cytoplasmaet av en oligopeptidtransporter kalt Ami, pluss en intracellulær transkripsjonsaktivator kalt ComR. Ifølge den foreslåtte modellen aktiveres ComR når denne aktivatoren binder til ComS*. I aktiv tilstand induserer ComR ekspresjonen av ComX ved å binde til comX promoteren. Ved å integrere comR genet og comX promoteren fusjonert til målgenet i genomet til S. pneumoniae ble vi i stand til å vise at systemet vårt fungerte etter hensikten. Vi brukte det essensielle licD1 genet, som er nødvendig for syntesen av teikoinsyre hos pneumokokker, til å prøve ut systemet vårt på et reelt biologisk problem. Resultatene viste at en gradvis reduksjon i mengden av LicD1 gav opphav til celler som var mye større og lengre enn normalt, noe som indikerer at pneumokokker med for lite teikoinsyre får problemer med å dele seg. Dette vellykkede resultatet viser at det titrerbare ekspresjonssystemet vi utviklet i artikkel III fungerer utmerket, og at det følgelig burde kunne bli et nyttig verktøy for studier av essensielle gener i pneumokokker og andre Gram-positive bakterier.